Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

BACKGROUND AND PURPOSE: Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress.

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

Evaluation of Intracellular Metabolism of Methotrexate in Hepatocytes and Embryonic Kidney Cells based on Folylpolyglutamate Synthetase and Gamma-Glutamyl Hydrolase Expression.

BACKGROUND: Methotrexate (MTX) is a common folic acid antagonist in clinical medicine, easily inducing a common adverse side effect of liver and kidney injury. It has been found that the expression of Folylpolyglutamate Synthetase (FPGS) and gamma-Glutamyl Hydrolase (GGH) may be closely related to that of related proteins to affect the intracellular metabolism of MTX. OBJECTIVE: The relationship between FPGS/GGH and MTXPGs accumulation in liver and kidney cells was explored by adjusting the expression of FPGS and GGH in cells using UPLC-MS/MS quantitative technology. METHOD: Based on UPLC-MS/MS quantitative techniques, the relationship between MTXPGs accumulation and FPGS/GGH in hepatocytes and embryonic kidney cells was explored by adjusting the expression of FPGS and GGH, and the effect of FPGS/GGH on the intracellular toxicity of MTX was comprehensively analyzed. RESULT: The results showed that the difference in methotrexate polyglutamates (MTXPGs) accumulation in liver and kidney cells was related to the difference in FPGS and GGH expression. The expression of FPGS interacted with that of GGH. These results suggest that the protein abundance ratio of FPGS to GGH (FPGS/GGH) has more potential to be used as a predictor of MTX efficacy than the FPGS or GGH single protein index. This can effectively avoid liver and kidney damage caused by MTX and guides the rational use of drugs in MTX. CONCLUSION: The results prove that there is a positive correlation between the FPGS/GGH and the accumulation of MTXPGS in liver and kidney cells. Summarily, the FPGS/GGH is expected to be a predictor for MTXPGs accumulation and provides an effective method to evaluate the toxicity caused by MTX.

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.

Interaction between glycolysis‒cholesterol synthesis axis and tumor microenvironment reveal that gamma-glutamyl hydrolase suppresses glycolysis in colon cancer.

Background: Metabolic reprogramming is a feature of cancer. However, colon cancer subtypes based on the glycolysis‒cholesterol synthesis axis have not been identified, and little is known about connections between metabolic features and the tumor microenvironment. Methods: Data for 430 colon cancer cases were extracted from The Cancer Genome Atlas, including transcriptome data, clinical information, and survival outcomes. Glycolysis and cholesterol synthesis-related gene sets were obtained from the Molecular Signatures Database for a gene set variation analysis. The relationship between the genomic landscape and immune landscape were investigated among four metabolic subtypes. Hub genes were determined. The clinical significance of candidate hub gene was evaluated in 264 clinical samples and potential functions were validated in vitro and in vivo. Results: Colon cancer cases were clustered into four metabolic subtypes: quiescent, glycolytic, cholesterogenic, and mixed. The metabolic subtypes differed with respect to the immune score, stromal score, and estimate score using the ESTIMATE algorithm, cancer-immunity cycle, immunomodulator signatures, and signatures of immunotherapy responses. Patients in the cholesterogenic group had better survival outcomes than those for other subtypes, especially glycolytic. The glycolytic subtype was related to unfavorable clinical characteristics, including high mutation rates in TTN, APC, and TP53, high mutation burden, vascular invasion, right colon cancer, and low-frequency microsatellite instability. GGH, CACNG4, MME, SLC30A2, CKMT2, SYN3, and SLC22A31 were identified as differentially expressed both in glycolytic-cholesterogenic subgroups as well as between colon cancers and healthy samples, and were involved in glycolysis‒cholesterol synthesis. GGH was upregulated in colon cancer; its high expression was correlated with CD4+ T cell infiltration and longer overall survival and it was identified as a favorable independent prognostic factor. The overexpression of GGH in colon cancer-derived cell lines (SW48 and SW480) inhibited PKM, GLUT1, and LDHA expression and decreased the extracellular lactate content and intracellular ATP level. The opposite effects were obtained by GGH silencing. The phenotype associated with GGH was also validated in a xenograft nude mouse model. Conclusions: Our results provide insight into the connection between metabolism and the tumor microenvironment in colon cancer and provides preliminary evidence for the role of GGH, providing a basis for subsequent studies.

Ultrasonically Enhanced ZD2767P-Carboxypeptidase G2 Deactivates Cisplatin-Resistant Human Lung Cancer Cells.

The prodrug-enzyme regimen ZD2767P+CPG2 is limited by low efficacy. Here, ultrasound was used to modulate ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against cisplatin-resistant human lung cancer cells. A549 and A549/DDP (resistant subline) cells received ZD2767P+CPG2 or ZD2767P+CPG2+US. Either ZD2767P+CPG2 or ZD2767P+CPG2+US led to cell death and apoptosis, and ZD2767P+CPG2+US produced stronger effects; comet assays revealed that these two means directly caused DNA double-strand break. Z-VAD-fmk and/or ferrostatin-1 increased the cell survival percentage, and Z-VAD-fmk decreased the apoptosis percentage. The level of transferrin was increased in treated cells, but those of ferroportin and glutathione peroxidase 4 (GPX4) were reduced, with higher intracellular levels of reactive oxygen species and of iron. Intracellular pharmacokinetics of ZD2767D (activated drug) indicated that the peak level, area under the drug level vs. time curve, and mean residence time in ZD2767P+CPG2+US were higher than those in ZD2767P+CPG2. Both ZD2767P+CPG2 and ZD2767P+CPG2+US were effective on xenograft tumors in nude mice; inhibitory rates were 39.7% and 63.5% in A549 tumors and 50.0% and 70.1% in A549/DDP tumors, respectively. A higher apoptosis level and a lower GPX4 level were noted in tumors receiving treatments. No severe adverse events were observed. These data demonstrated that ZD2767P+CPG2+US deactivated cancer cells via apoptosis and ferroptosis pathways, being a candidate therapy for cisplatin-resistant lung cancer.

Carboxypeptidase G and pterin deaminase metabolic pathways degrade folic acid in Variovorax sp. F1.

BACKGROUND: Folic acid (FA) is a synthetic vitamin (B9) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved. RESULTS: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts. CONCLUSION: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.

Seeing the unseen: a trifoliate (MYB117) mutant allele fortifies folate and carotenoids in tomato fruits.

In tomato (Solanum lycopersicum), mutations in the gene encoding the R2R3-MYB117 transcription factor elicit trifoliate leaves and initiate the formation of axillary meristems; however, their effects on fruit ripening remain unexplored. The fruits of a new trifoliate (tf) mutant (tf-5) were firmer and had higher °Brix values and higher folate and carotenoid contents. The transcriptome, proteome, and metabolome profiling of tf-5 reflected a broad-spectrum change in cellular homeostasis. The tf-5 allele enhanced the fruit firmness by suppressing cell wall softening-related proteins. tf-5 fruit displayed a substantial increase in amino acids, particularly γ-aminobutyric acid, with a parallel reduction in aminoacyl-tRNA synthases. The increased lipoxygenase protein and transcript levels seemingly elevated jasmonic acid levels. In addition, increased abscisic acid hydrolase transcript levels coupled with reduced precursor supply lowered abscisic acid levels. The upregulation of carotenoids was mediated by modulation of methylerythreitol and plastoquinone pathways and increased the levels of carotenoid isomerization proteins. The upregulation of folate in tf-5 was connoted by the increase in the precursor p-aminobenzoic acid and transcript levels of several folate biosynthesis genes. The reduction in pterin-6-carboxylate levels and γ-glutamyl hydrolase activity indicated that reduced folate degradation in tf-5 increased folate levels. Our study delineates that in addition to leaf development, MYB117 also influences fruit metabolism. The tf-5 allele can be used to increase γ-aminobutyric acid, carotenoid, and folate levels in tomato.

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa.

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation-putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels.

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.

Quantification of folate in food using deconjugase of plant origin combined with LC-MS/MS: A method comparison of a large and diverse sample set.

A round robin comparison was performed in order to test the performance of a recently developed LC-MS/MS method for quantification of 6 folate forms. Eighty-nine samples representing the food groups of fruits, vegetables, legumes, cereals, dairy products, meat, and offal were analyzed by two LC-MS/MS methods and a microbiological assay (MA). A plant-origin deconjugase enzyme (Arabidopsis thaliana) for deconjugation of folates (PE-LC-MS/MS), or animal-origin deconjugase (rat serum and chicken pancreas) (AE-LC-MS/MS) was used in the LC-MS/MS methods, each in a single enzymatic step. In contrast, the MA involved tri-enzyme extraction including human plasma as a deconjugase. A significant bias of 17% lower and 25% higher results was found when PE-LC-MS/MS was compared to MA and AE-LC-MS/MS, respectively. The PE-LC-MS/MS provides fast quantification of various folate vitamers and total folate content, which could be a proper substitute to the currently standardized but imprecise and time-consuming microbiological assay in the future.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule.

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs -out of 1155- with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

Growth Response of Lactobacillus rhamnosus Chloramphenicol-Resistant Strain ATCC 27773 to Pteroyl-Mono- and -Di-Glutamates.

This study investigated the effect of various concentrations of pteroyl-mono-γ-glutamate (PteGlu1) and pteroyl-di-γ-glutamate (PteGlu2) on the growth of Lactobacillus rhamnosus strains ATCC 7469 (wild-type strain) and ATCC 27773 (chloramphenicol-resistant strain) used for folate microbiological assays. At concentrations of 0.025-0.20 nmol/L, the growth of the chloramphenicol-resistant strain was stimulated to a greater extent by PteGlu1 than by PteGlu2, but the wild-type strain did not show such phenomena. L. rhamnosus ATCC 27773 bioassays were used to determine the total folate content of various foods treated with a chicken pancreas folate conjugase. This showed a significantly lower value when PteGlu1 was used as a calibrator than with PteGlu2. These results indicated that PteGlu2 should be the standard folate compound when chicken pancreas folate conjugase is used in preparing samples for L. rhamnosus ATCC 27773 bioassay.

2,4-Diamino-N10-methylpteroic acid (DAMPA) crystalluria in a patient with osteosarcoma treated with carboxypeptidase-G2 rescue after high-dose methotrexate-induced nephrotoxicity.

BACKGROUND: High-dose methotrexate (HDMTX) therapy is a key component of many chemotherapy protocols. However, some patients develop HDMTX-induced nephrotoxicity. Carboxypeptidase-G2 (CPDG2) hydrolyses MTX into 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamic acid, and is used as a rescue agent in patients with nephrotoxicity and delayed elimination. Despite the frequency of HDMTX-induced renal injury, crystalluria is uncommon. Furthermore, crystals are rarely identified by conventional chemical methods. OBJECTIVE: To determine the composition of crystalluria in a patient with osteosarcoma who was treated with CPDG2. METHODS: Crystalluria was evaluated by optical microscopy, and chemical identification was performed by Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and Orbitrap™ high-resolution mass spectrometry (HRMS). RESULTS: The HRMS spectra of the patient's urine sediment showed a main peak at m/z 326.13, corresponding to the molecular mass of DAMPA [(C15H15O2N7) + H+]. The FT-IR spectral patterns of the sediment and DAMPA were not identical. SEM was unable to identify the crystal. CONCLUSION: DAMPA crystalluria was identified by Orbitrap™ HRMS in a patient treated with CPDG2 after HDMTX nephrotoxicity. This case reinforces the need to implement adequate measures to prevent nephrotoxicity. In cases of HDMTX-induced nephrotoxicity, urine sediment analysis should be requested.

Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase.

The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53­null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild­type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.

High-Level γ-Glutamyl-Hydrolase (GGH) Expression is Linked to Poor Prognosis in ERG Negative Prostate Cancer.

γ-glutamyl-hydrolase (GGH) is a ubiquitously-expressed enzyme that regulates intracellular folate metabolism for cell proliferation, DNA synthesis, and repair. Employing GGH immunohistochemistry on a tissue microarray with 12,427 prostate cancers, we found that GGH expression was negative to low in normal prostate epithelium, whereas 88.3% of our 10,562 interpretable cancers showed GGH expression. GGH staining was considered as low intensity in 49.6% and as high intensity in 38.6% of cancers. High GGH expression was linked to the TMPRSS2:ERG-fusion positive subset of cancers (p < 0.0001), advanced pathological tumor stage, and high Gleason grade (p < 0.0001 each). Further analysis revealed that these associations were merely driven by the subset of ERG-negative cancers, High GGH expression was weakly linked to early biochemical recurrence in ERG negative cancers (p < 0.0001) and independent from established histo-pathological parameters. Moreover, GGH expression was linked to features of genetic instability, including presence of recurrent deletions at 3p, 5q, 6q, and 10q (PTEN, p ≤ 0.01 each), as well as to accelerated cell proliferation as measured by Ki67 immunohistochemistry (p < 0.0001). In conclusion, the results of our study identify GGH as an ERG subtype specific molecular marker with modest prognostic relevance, which may have clinical relevance if analyzed in combination with other molecular markers.